표제지
목차
Abstract 6
I. 서론 8
II. 재료 및 방법 12
1. 실험균주 12
1-1. 환경수 채취 12
1-2. Staphylococci의 분리 13
2. 약제 감수성 실험 16
3. PCR 17
3-1. Total DNA 분리 17
3-2. Primer 제작 18
3-3. PCR 21
4. 클로닝 22
4-1. Gel elution 22
4-2. 클로닝 22
5. 플라스미드 분리 23
6. Conjugation 24
7. Transposon의 구조 유전자적 분석 25
8. tet(M)의 계통발생학적 분석 30
III. 결과 31
1. 해양환경 내의 Staphylococci 균주 분포 31
2. Staphylococci의 항생제 내성 특성 분석 34
3. PCR에 의한 내성 유전자 확인 40
4. 내성 유전자의 전이 특성 분석 46
5. tet(M)과 transposon의 연관성 확인 48
6. Transposon에 위치한 ORF의 확인 51
7. tet(M)의 유전자적 상동성과 Mosaic 구조 분석 54
8. tet(M)의 upstream 부위의 염기서열 비교 63
9. Staphylococci의 tet(M)의 계통발생학적 분석 67
IV. 고찰 69
V. 요약 89
VII. 참고 문헌 95
Table 1. PCR Primers for Staphylococci 15
Table 2. PCR Primers for detection of the various tet genes 19
Table 3. PCR Primers for erm genes 20
Table 4. PCR Primers for determination of the int gene indicated transposon 28
Table 5. PCR Primers for flanking region of tet(M) 29
Table 6. Number of Staphylococci isolates in environmental water 32
Table 7. The number of the multiple-antibiotic-resistant isolates from environmental water 37
Table 8. Genomic characterization of Tc or Em-resistant Staphylococci from environment and human. 43
Table 9. Genomic characterization of Tc and Em-resistant Staphylococci from environment and human. 44
Table 10. Isolated Tc-resistant strains used in this study 45
Table 11. Transfer of Resistant genes to recipient and transconjugant 47
Table 12. Determination of the ORF in gene structure of transposon for determinate to location of tet(M) from each sources 52
Table 13. Homology of the tet(M) gene from each source by comparing with several transposon as reference 56
Fig. 1. Classification of the Staphylococci included Staphylococcus spp. and S. aureus by using BPA plate and coagulase test. 14
Fig. 2. Schematic structures of different transposon carrying tetracycline resistant gene, tet(M) and other genes. The black arrow box indicates that it was different with sequence of the same ORF on the other transposon. 27
Fig. 4. Proportion of Staphylococci from environmental water. 33
Fig. 5. Proportion of the antibiotics-resistant isolates about different antibiotics. 36
Fig. 6. Proportion of the multi-resistant Staphylococci isolates. S means susceptible, and R1~6 designate the number of antibiotics that exhibit resistance to Staphylococci isolate. 38
Fig. 7. Proportion of the Tc-resistant Staphylococci (Staphylococcus spp., n=35; S. aureus, n=9) from environmental water. As Staphylococci from human, only a single strain, S. aureus (n=50) was used. Each of the MIC value with tetracycline was determined by using broth dilution... 39
Fig. 8. Agarose gel electrophoresis of amplicons generated by PCR of tet(M) or tet(K). Lane, 1, 2, 3, 4, 5, 6, tet(M) from environmental isolates; Lane, 7, 8, tet(K) from environmental isolates; Lane, 9, 10,... 42
Fig. 9. Agarose gel electrophoresis of amplicon generated by PCR of int gene. Lane, 1, 2, 3, 4, 5, 6, tet(M) from environmental isolates; Lane, 7, Streptococcus spp. K8 as positive control; Lane, 8, Negative control for int-1,2 primer set; Lane, 9, 10, tet(M) from human isolates; Lane 11,... 50
Fig. 10. (A) Schematic structure of the Tn916/1545 for design specific primer used to determination of location of tet(M). The arrow designated to location of the primer on transposon. Each amplicon was shown in panel (B) below. Tn5801-specific TMup amplicon (asterisk) was shown... 53
Fig. 11. Comparison analysis of the DNA nucleotide full sequence of tet(M) between environmental or human isolates and other transposon as reference from GenBank (NCBI). 57
Fig. 12. Comparison of the mosaic structures of tet(M) in Tc resistant Staphylococci from environment and human. slashed, black and open bars indicate regions showing high nucleotide sequence homology with those in tet(M) on Tns, 1545, 916 and 5801, respectively. New... 62
Fig. 13. Comparison analysis of the 5' of the tet(M) including promoter sequence. The promoter region (asterisk) was referred to a report from Su et al. (1992). The arrow indicate the direction of start and stop codon that encodes ORF, and vertical bar means stop codon of the ORF. 65
Fig. 14. Phylogenetic tree constructed based on full sequences of tet(M). The tree was platted using the neighbor-joining(N-J) method. 68